Renal and Cardiovascular Protocol
PhysGen
I. Experimental setup for renal and cardiovascular
studies in conscious rats (instrumentation and
calibration procedures)
Instrumentation and equipment used in setup [order information listed in section V]:
· Surgical station: thermostatically controlled surgical board, stereo-microscope, fiber-optic light, surgical instrument pack, recovery station with temperature controlled pads, HEPA filter hood area for preparation of animals for surgery. [Please see the Surgical Pack Protocol section where complete details are given related to the instruments, supplies, and equipment used in the surgical preparation]
· Chronic Monitoring Facility—recording rooms used for studies allow simultaneous collection of hemodynamic data from up to 64 animals. Specially designed cages permit collection of urine for analysis. [A CMF section will be added at a later date]
· Biochemical Core Lab- analysis of samples collected for electrolytes, protein, plasma renin activity, osmolality, and creatinine is performed in the PGA Biochemistry Core Lab and the Physiology Biochemical Core Laboratory [See Biochemistry Protocol section for more complete description]
The following series of pictures depict the experimental set-up as used daily.
II. Experimental protocol for renal and cardiovascular studies.
A. Surgical preparation of animals for study.
1. Carefully don sterile gloves in the appropriate size. Once these gloves are on, do not touch any non-sterile surfaces! Doing so will compromise the sterile surgery and, consequently, the rat’s health. If at any time you do accidentally touch a non-sterile area or find a hole in your glove, replace the glove immediately.
2. Arrange the sterile instruments so that they are organized and within easy reach, taking care that they at all times remain in the sterile field. Attach the 22-gauge adapter to the sterile 1cc syringe and fill with sterile saline. Use this to fill the microrenathane catheter you will implant during the surgery. Check again to insure the rat is adequately anesthetized before you make your incision!
3. Implanting the catheter: The femoral vein and artery run along the same path as the femur. The incision should be made in that area and close to, but not on, the abdominal wall. It is not advisable to make a large incision, as these seem to irritate the rats more readily, but the incision needs to be large enough to work comfortably (about 1 to 1.5 cm should be sufficient). Once the skin is open, look through the microscope to continue with the rest of the implantation.
a. Using the micro-dissecting forceps, carefully part the tissues as you tunnel down toward the femoral artery and vein. The actual implantation will occur very close to the abdominal wall, so work in that area. Avoid tearing the numerous small blood vessels in the connective tissue, the less disturbance caused the better. When you encounter a rather large ‘knot’ of the biggest vessels you have seen so far, you are near to your goal. The area used for implantation is located between this ‘knot’ and the abdominal wall. As you carefully part the clear connective tissue in this area, the ‘knot’ will move away and the straight section of femoral vein and artery often hidden by it, will be exposed. The vein is the larger, purple vessel, and the artery is the thinner white one.
b. Next you will separate the artery from the vein. IMPORTANT: You must avoid disturbing the numerous nerves which run beside the artery. They appear white and thready. Once manipulated, the nerves do not recover, and will adversely effect the rat’s ability to use the limb, to the point that the rat will mutilate it’s own leg, not recognizing it as his own! For best results, work between the artery and vein to separate the two, and avoid touching the side near the nerves altogether.
c. When the artery has been isolated, carefully and gently lift it with the forceps and draw three ties beneath it, taking care not to damage the vessel in the process. Use the microscope to arrange the ties so that one is at the distal end of the cleared vessel, one is in the center, and the third is at the proximal end. The tie at the distal end should be as close to the end of the cleared section of artery as possible. Tie this one in one (tight) knot. This will occlude the vessel. Attach a hemostat to the free ends of this tie so that a constant, but not excessive, tension is kept. With the tie at the proximal end, start a knot, draw it down until the opening is still large enough to allow the catheter to pass through, making sure it is as close to the abdominal wall as the cleared vessel will allow. Attach another hemostat to the free ends of this tie to achieve constant (but again, not excessive) tension to the vessel. Start a knot in the center tie, leaving it just as open as the previous one. Do not attach a hemostat to this center tie, but arrange it close to the top tie and leave the ends loose.
d. At this point, you will need to measure the catheter’s tip for a proper fit. Without allowing the catheter to touch the rat, determine the length the tip should be to reach from the vessel incision site to the point of, but not into, the aorta.
e. You are now ready to make your cut into the artery. Check to make sure the top tie is effectively occluding the vessel! Trim the tip to that distance, creating a slight bevel on the very end. Take care that the bevel is not too sharp, as it will easily puncture the vessel wall during implantation. Recheck the entire catheter to insure it is completely filled with saline and there are no air bubbles present, and apply a catheter occluding forceps just beyond the syringe.
f. Using the Vannas scissors carefully cut into the artery wall close to the distal tie, but allowing for space to pass the catheter over the tie. Needless to say, do not cut through the artery! It is better to have to cut twice in the same spot than to cut too much. Do not cut more than halfway through the vessel, especially when working with a Dahl rat. If, while you are making the cut, your field of view is suddenly filled with blood do not panic. Quickly put more tension on the upper tie by simply moving the hemostat back a little, effectively stopping the flow. Clean up the blood thoroughly, as it is an irritant to tissues outside of the vessels.
g. With the artery incised, you are now ready to insert the catheter. Using the Dumont micro dissecting forceps, hold the tip of the catheter in one (allowing enough of the tip to protrude to begin the insertion into the artery) and with the other, carefully lift the top of the incision in the artery. Note: Avoid pulling excessively on this incision, as oftentimes it will enlarge and will eventually reach the upper tie making it extremely difficult, if not impossible, to successfully place the catheter. Always be aware that these forceps have sharp, pointed ends. Take care that they do not puncture the vessels or damage the nerves as you work.
h. Holding the top lip of the incision, carefully insert the catheter tip into the opening. When enough of the tip has been implanted to allow it, release the lip of the incision and lightly grip the vessel around the catheter. This will allow you to hold the vessel in place as you continue to thread the catheter into the artery. Try to keep the artery parallel to the vein and as close to it’s natural position as possible. This will reduce the chance of punctures, and twisting of the vessel. When the tip has passed the middle tie and is near to, but not touching, the top tie, continue to hold the catheter inside the vessel while you use the other forceps to loosen the top tie. This can usually be accomplished by simply pulling down a little, moving the hemostat enough to allow the tie to be slack. Carefully pass the catheter tip through the top tie and continue advancing until the base of the catheter tip (where it joins the larger body of the catheter) rests at the incision in the artery.
i. Move the center tie as close as possible to the catheter’s joint, and use it to secure the vessel to the catheter tip at the base. Tie a double knot, but keep in mind that it is possible to occlude the catheter by tying too tightly! (Check the catheter after each knot is made to insure it is still working properly. If not, loosen the offending knot and retie.) Remove the hemostat from the top tie and tighten the knot between the center tie and the abdominal wall. Remove the hemostat from the bottom tie and secure this tie to the catheter behind the joint in the catheter, on the larger tubing (If this tie is not on the larger tubing, the catheter will easily pull out of the vessel). This knot can and should be tied rather tightly. Check the catheter once more to insure proper function then insert a 22-gauge plug into the end of the catheter. Trim all tie ends close to the knots, taking care to avoid cutting the knots in the process.
j. Apply a small amount of Vet Bond to the site where the catheter joint meets the vessel. Take care that no Vet Bond touches any other surface. Vet Bond dries very quickly and is irritating to the rat is too much is used.
k. Next, fill the cavity with antibiotic ointment (neomycin & polymyxin B sulfate, bacitracin zinc & hydrocortisone acetate).
l. Using scissors as a spreader, tunnel a path for the trochar just below the skin, beginning at the top of the incision in the skin until the scissors can reach no further. Carefully thread the trochar from the incision to a location about 2 cm from the scapulas on the animal’s back. While advancing the trochar, be sure to keep the tip of the bevel against the underside of the skin to avoid damaging muscles, vital organs, etc. When the site has been reached, leave the trochar in place and apply betadine to the site. Using scissors, create an opening large enough for the trochar, and later the button, to pass through. Leaving the trochar protruding from the opening at the shoulders and at the incision in the leg, prepare the catheter by wiping the entire exposed length with alcohol. Without letting the catheter touch anything else, carefully thread the catheter end into the trochar at the leg and advance it until it appears at the shoulders. At this point, observe the catheter within the leg as you continue slowly pulling it at the shoulders. Make sure the catheter in the leg does not twist or pull the artery, and leave a slight loop there so that it does not pull the artery as the catheter is later manipulated.
m. When the catheter is correctly in place, carefully remove the trochar by pulling gently on the end protruding at the shoulders. When the trochar is completely out, check the leg again to insure that the catheter did not move.
n. Now the leg incision can be closed, using Braunamide suture and interrupted stitches. Be sure to completely close the incision and cut ends as short as possible to minimize the opportunity for the rat to open the incision. Apply Betadine to the closed incision.
4. Implanting the button: Reposition the rat so that he is on his chest and facing you. Using scissors, enlarge the area under the skin surrounding the shoulder incision so that the button will rest upright and flat when the surgery is completed. Apply antibiotic ointment liberally to the area just enlarged with the scissors. Thread the catheter end through the button and bring the button to rest on the incision. Using forceps, work the outer edge of the button under the skin until all of the dacron is below the skin and lying flat. Tack the button into place with silk suture, passing the needle through the skin, then the dacron, then back out through the skin close to the initial entry site, and tie off. Do this in four evenly spaced sutures. Use Braunamide suture to close the skin incision, securing the button. Next, thread the catheter through the spring and carefully push the spring end into the button tubing until it almost touches the incision. Use a piece of tape to secure the other end of the spring to the overhead lamp, then apply Super Glue Gel to the very top of the button, continue applying the glue to about 1/2” of the spring above it. Allow it to dry thoroughly.
5. Recovery: Administer injectable antibiotic (Penicillin G) at 0.10cc/100 gm bwt I.M. to the right leg. Return rat to his recovery cage that you had placed on the warming pad in the recovery area. Make sure the spring is protruding through the wire bar lid to prevent destruction of the catheter by the rat during recovery. Observe the rat frequently during recovery. Each time, manipulate the leg with the implanted catheter to aid collateral circulation and flexion of the limb. Massage the foot and “bicycle” the leg several times during recovery. When the rat is sternal and moving about the cage, administer Buprenex 0.05cc/100 gm bwt s.c. When the rat has completely recovered from the effects of the Buprenex, he can be taken to his cage in the recording room. The rat must be conscious enough to drink from the water bottle upon return from the home cage.
6. Daily assessment of animal health:Throughout the remainder of the protocol,each rat must be observed for signs of illness or injury. First thing each morning, carefully examine each rat. Simply looking through the cage wire is not sufficient. Open the cage and watch the rat as he moves about. Note and record observations for each of the following: